Method of treating malignancy associated hypercalcemia using active vitamin D analogues

ABSTRACT

Methods utilizing active vitamin D analogs for the treatment of malignancy-associated hypercalcemia. Methods comprise the application of an effective amount of a hypocalcemic vitamin D compound to alleviate hypercalcemia, lower serum parathyroid hormone related protein (PTHrP) levels.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/596,149 filed Feb. 23, 1998, which is a continuation-in-partof U.S. application Ser. No. 08/781,910, filed Dec. 20, 1996, now U.S.Pat. No. 5,763,429, all of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not Applicable

BACKGROUND OF THE INVENTION

[0003] This invention relates generally to a method of treatingmalignancy-associated hypercalcemia (MAH), and in particular, to the useof active forms of vitamin D to reduce hypercalcemia associated withinhibit the hyperproliferative diseases.

[0004] Extensive research during the past two decades has establishedimportant biologic roles for vitamin D apart from its classic role inbone and mineral metabolism. Specific nuclear receptors for1α,25-dihydroxyvitamin D₃, the hormonally active form of vitamin D, arepresent in cells from diverse organs not involved in calciumhomeostasis. For example, specific, biologically active vitamin Dreceptors have been demonstrated in the human prostatic carcinoma cellline, LNCaP, (Miller et al., 52 Cancer Res. (1992) 515-520); Vitamin Dreceptors have also been described for many other neoplastic cells,e.g., carcinomas of the breast and carcinomas of the colon.

[0005] It has been reported that certain vitamin D compounds andanalogues are potent inhibitors of malignant cell proliferation and areinducers/stimulators of cell differentiation. For example, U.S. Pat. No.4,391,802 issued to Suda et al. discloses that 1α-hydroxyvitamin Dcompounds, specifically 1α,25-dihydroxyvitamin D₃ and 1α-hydroxyvitaminD₃, possess potent antileukemic activity by virtue of inducing thedifferentiation of malignant cells (specifically leukemia cells) tononmalignant macrophages (monocytes), and are useful in the treatment ofleukemia. Antiproliferative and differentiating actions of1α,25-dihydroxyvitamin D₃ and other vitamin D₃ analogues have beenreported with respect to cancer cell lines. More recently, anassociation between vitamin D receptor gene polymorphism and cancer riskhas been reported, suggesting that vitamin D receptors may have a rolein the development, and possible treatment, of cancer.

[0006] These previous studies have focused exclusively on vitamin D₃compounds. Even though these compounds may indeed be highly effective inpromoting differentiation in malignant cells in culture, their practicaluse in differentiation therapy as anticancer agents is severely limitedbecause of their equally high potency as agents affecting calciummetabolism. At the levels required in vivo for effective use as, forexample, antileukemic agents, these same compounds can induce markedlyelevated and potentially dangerous blood calcium levels by virtue oftheir inherent calcemic activity. That is, the clinical use of1α,25-dihydroxyvitamin D₃ and other vitamin D₃ analogues as anticanceragents is precluded, or severely limited, by the risk of hypercalcemia.

[0007] Hyperalcemia is frequently associated with malignancy (MAH), andis often a major contributor to morbidity and complicates clinicalmanagement of the malignancy. Parathyroid hormone related protein(PTHrP) is closely related to parathyroid hormone (PTH) and binds to thesame receptor as PTH as well as other receptors. PTHrP is one of themain causative substances of such hypercalcemia, and is overproduced bymalignant cells. 1,25-dihydroxyvitamin D₃ has been found to repress thetranscription of the PTHrP gene in cells, however, the1,25-dihydroxyvitamin D₃ compounds themselves increase serum calciumlevels. Therefore a need exists for compounds with greater specificactivity and selectivity of action, i.e., vitamin D compounds withantiproliferative and differentiating effects but which have lesscalcemic activity.

BRIEF SUMMARY OF THE INVENTION

[0008] The present invention provides a method of treatingmalignancy-associated hypercalcemia (MAH) such as that associated withhyperproliferative cell growth and/or abnormal cell differentiation. Themethod includes use of active vitamin D compounds to treat hypercalcemiaand reduce serum parathyroid hormone related protein (PTHrP) levels.

[0009] The foregoing, and other advantages of the present invention, arerealized in one aspect thereof in a method of treatingmalignancy-associated hypercalcemia from the hyperproliferative activityof human neoplastic or hyperplastic cells, comprising treating the cellswith an effective amount of a hypocalcemic hydroxyvitamin D compoundhaving a hydrocarbon moiety substituted at the C-24 position on thesidechain of the molecule. The treating step includes inhibitingproliferation of, and inducing and enhancing differentiation in suchcells.

[0010] A hydroxyvitamin D compound in accordance with the presentinvention is an active vitamin D and is suitably represented by theformula (I) described hereafter. Suitable compounds of formula (I) are1α,24-dihydroxyvitamin D₂, 1α,24-dihydroxyvitamin D₄,1α,25-dihydroxyvitamin D₄, 1α,25-dihydroxyvitamin D₂, 1α-hydroxyvitaminD₂ and 1α-hydroxyvitamin D₄.

[0011] The effective or therapeutic amount of the hypocalcemichydroxyvitamin D compounds administrable in accordance with the presentinvention to patients in need on a daily basis per kilogram of bodyweight ranges from 0.01 μg/kg/day to 2.0 μg/kg/day.

[0012] In another aspect of the invention, lowering serum parathyroidhormone related protein (PTHrP) levels in patients suffering fromhypercalcemia is accomplished by a method comprising, administering tothese patients an effective amount of a hypocalcemic vitamin D compound,to lower the serum parathyroid hormone related protein (PTHrP) level.

[0013] The hypocalcemic vitamin D compounds are also valuable for thetreatment of breast, prostate and colon cancer, as well as otherneoplasms such as pancreatic cancer, endometrial cancer, testicularcancer, small cell and non-small cell cancer of the lung (includingsquamous, adneocarcinoma and large cell types), squamous cell of thehead and neck, bladder, ovarian and cervical cancers, myeloid andlymphocyltic leukemia, lymphoma, hepatic tumors, medullary thyroidcarcinoma, multiple myeloma, retinoblastoma, and sarcomas of the softtissue and bone, i.e. neoplasms that express a vitamin D receptor.

[0014] In accordance with the present invention, when effective amountsof the hypocalcemic vitamin D compounds are administered to patientswith MAH, significantly redeuced hypercalcemia is observed than isobserved after the same amount of an activated vitamin D₃ (e.g., 1α-OHD₃, 1α,25-(OH)₂ D₃) is administered in previously known formulations.Thus, the compound in accordance with the present invention has animproved therapeutic index relative to active forms of vitamin D₃analogues.

[0015] Accordingly, another aspect of the invention is a method oftreating malignancy associated hyercalcemia comprising administering toa subject who is suffering therefrom an effective amount of activevitamin D compound which has, or attains through metabolism in vivo, avitamin D receptor (VDR) binding affinity substantially equivalent tothe binding affinity of 1α,25-dihydroxyvitamin D₃ and has ahypercalcemia risk substantially lower that that of1α,25-dihydroxyvitamin D₃, to normalize or reduce serum calcium levels.

[0016] For treatment for malignancy-associated hypercalcemia and theunderlying malignant condition in accordance with the present invention,the active vitamin D is suitably administered alone as an activeingredient in a pharmaceutical composition, or is co-administered withan anticancer agent.

[0017] Further, included within the scope of the present invention isthe co-administration of a hypocalcemic vitamin D compound with acytotoxic or anticancer agent. Such agents suitably includeantimetabolites (e.g., 5-fluoro-uracil, methotrexate, fludarabine),antimicrotubule agents (e.g., vincristine, vinblastine, taxanes such aspaclitaxel, docetaxel), an alkylating agent (e.g., cyclophasphamide,melphalan, biochoroethylnitrosurea, hydroxyurea), platinum agents (e.g.cisplatin, carboplatin, oxaliplatin, JM-216, CI-973), anthracyclines(e.g., doxrubicin, daunorubicin), antibiolitics (e.g., mitomycin,idarubicin, adriamycin, daunomycin), topoisomerase inhibitiors (e.g.,etoposide, camptothecins) or any other antineoplastic agents.(estramustine phosphate, prednimustine).

[0018] It is anticipated that the active vitamin D compounds used incombination with various anticancer drugs can give rise to asignificantly enhanced cytotoxic effect on cancerous cells, thusproviding an increased therapeutic effect. Specifically, as asignificantly increased growth-inhibitory effect is obtained with theabove disclosed combinations utilizing lower concentrations of theanticancer drugs compared to the treatment regimes in which the drugsare used alone, there is the potential to provide therapy whereinadverse side effects associated with the anticancer drugs areconsiderably reduced than normally observed with the anticancer drugsused alone in larger doses. Possible dose ranges of theseco-administered anticancer agents are about 0.1 to 20 mg/kg/day.

[0019] Also included within the scope of the present invention is theco-administration of effective dosages of a hypocalcemic vitamin Dcompound in conjunction with administration of hormones or other agents,e.g., estrogens, which are known to ameliorate bone diseases ordisorders. For example, prostate cancer often metastasizes to bone,causing bone loss and associated pain. Such bone agents may includeconjugated estrogens or their equivalents, calcitonin, bisphosphonates,calcium supplements, cobalamin, pertussis toxin and boron.

[0020] In another aspect, the invention is a pharmaceutical compositionwhich includes an anticancer agent which is an active hypocalcemicvitamin D compound; an agent selected from the group consisting of (i)an anticancer agent, (ii) a bone agent, and combinations thereof; and aphysiologically acceptable carrier.

[0021] Other advantages and a fuller appreciation of specificadaptations, compositional variations, and physical attributes will begained upon an examination of the following detailed description ofpreferred embodiments, taken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWING(S)

[0022] Not Applicable

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides an effective method for thetreatment of hypercalcemia, i.e. unphysiologically high and deleteriousblood calcium levels, associated with neoplastic and hyperproliferativediseases. Particularly, the present invention relates to therapeuticmethods for ameliorating or alleviating the hypercalcemia associatedwith the hyperproliferative cellular activity of malignant andneoplastic diseases, as well as inducing, enhancing or promoting celldifferentiation in the diseased cells. The present invention provides anovel treatment of a patient suffering from a hyperproliferative diseasewith an active hypocalcemic vitamin D compound. Preferably, the activevitamin D analogue is a hydroxyvitamin D compound and is suitablyrepresented by formula (I) as described hereinbelow. The active vitaminD analogue is provided to the patient without itself causingdose-limiting hypercalcemia and hypercalciuria, and in fact, reduces thehypercalcemia caused by the malignancy. These attributes are achievedthrough specific chemical properties of the hypocalcemic vitamin Dcompounds as described.

[0024] In accordance with the present invention, when effective amountsof the hypocalcemic active vitamin D compounds are administered topatients with malignant diseases, the hypercalcemia is reduced, thePTHrP serum level is reduced, and the proliferative activity of theabnormal cells is inhibited, redeuced, or stabilized, and celldifferentiation is induced, promoted or enhanced. Thus, the hypocalcemicvitamin D compounds of the present invention have an improvedtherapeutic index relative to active forms of vitamin D₃ analogues.

[0025] It is known that vitamin D₃ must be hydroxylated in the C-1 andC-25 positions before it is activated, i.e., before it will produce abiological response. A similar metabolism appears to be required toactivate other forms of vitamin D, e.g., vitamin D₂ and vitamin D₄.Therefore, as used herein, the term “activated vitamin D” or “activevitamin D” is intended to refer to a vitamin D compound or analogue thathas been hydroxylated in at least the C-1, C-24 or C-25 position of themolecule and either the compound itself or its metabolites in the caseof a prodrug, such as 1α-hydroxyvitamin D₂, binds the vitamin D receptor(VDR). For example, “prodrugs” are vitamin D compounds which arehydroxylated in the C-1. Such compounds undergo further hydroxylation invivo and their metabolites bind the VDR.

[0026] The term “hypocalcemic vitamin D compound” is in reference toactive vitamin D analogs which demonstrate hypocalcemic activity, i.e.have low calcemic activity relative to that of 1α,25-dihydroxyvitaminD₃, including 24-hydroxyvitamin D compounds, 25-hydroxyvitamin compoundsand I a-hydroxyvitamin compounds.

[0027] Also, as used herein, the term “lower” as a modifier for alkyl,alkenyl acyl, or cycloalkyl is meant to refer to a straight or branched,saturated or unsaturated hydrocarbon radical having 1 to 4 carbon atoms.Specific examples of such hydrocarbon radicals are methyl, ethyl,propyl, isopropyl, butyl, isobutyl, t-butyl, ethenyl, propenyl, butenyl,isobutenyl, isopropenyl, formyl, acetyl, propionyl, butyryl orcyclopropyl. The term “aromatic acyl” is meant to refer to aunsubstituted or substituted benzoyl group.

[0028] As used herein, the term “hydrocarbon moiety” refers to a loweralkyl, a lower alkenyl, a lower acyl group or a lower cycloalkyl, i.e.,a straight or branched, saturated or unsaturated C₁-C₄ hydrocarbonradial.

[0029] The compound in accordance with the present invention is anactive hypocalcemic vitamin D compound. The active vitamin D provided issuch that the compound has a hydrocarbon moiety at the C-24 position,e.g. a lower alkyl, alkenyl or acyl group as the C-24 position. Further,the active vitamin D in accordance with the present invention may havean unsaturated sidechain, e.g., there is suitably a double bond betweenC-22 and C-23, between C-25 and C-26 or between C-26 and C-27.

[0030] The hypocalcemic hydroxyvitamin D of the present inventionsuitably has the general formula described in formula (I)

[0031] wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹and R² cannot both be alkenyl, or taken together with the carbon towhich they are bonded, form a C₃-C₈ cyclocarbon ring; R³ is lower alkyl,lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl,O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X¹is hydrogen or hydroxyl, X² is hydrogen or hydroxyl, or, may be takenwith R¹ or R², to constitute a double bond, and X³ is hydrogen orhydroxyl provided that at least one of X¹, X², or X³ is hydroxyl, and Yis a methylene group if the bond to Y is a double bond or is a methylgroup or hydrogen if the bond to Y is a single bond.

[0032] A 1α-hydroxyvitamin D compound of formula (I) is characterized bythe general formula (II):

[0033] wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹and R² cannot both be an alkenyl, or taken together with the carbon towhich they are bonded, form a C₃-C₈ cyclocarbon ring; R³ is lower alkyl,lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl,O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X¹is hydrogen or hydroxyl, X² is hydrogen or hydroxyl, or, may be takenwith R¹ or R², to constitute a double bond, and Y is a methylene groupif the bond to Y is a double bond or is a methyl group or hydrogen ifthe bond to Y is a single bond.

[0034] Specifically, 1α-hydroxyvitamin D compounds in accordance withthe present invention are characterized by the general formula (III):

[0035] wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹and R² cannot both be an alkenyl, or taken together with the carbon towhich they are bonded, form a C₃-C₈ cyclocarbon ring; R³ is lower alkyl,lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl,O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X¹is hydrogen or hydroxyl, and X² is hydrogen or hydroxyl, or, may betaken with R¹ or R², to constitute a double bond.

[0036] The hypocalcemic hydroxyvitamin D compounds of the presentinvention are those that have effective antiproliferative and celldifferentiation activity (i.e., reversal of malignant transformation),but have a lower tendency or inability to cause hypercalcemia and/orhypercalciuria i.e. they are hypocalcemic compounds that have lowcalcemic activity relative to that of 1α,25-dihydroxyvitamin D₃. Inother words, the compounds of the present invention can be administeredat dosages that allow them to act as antiproliferative agents and celldifferentiation agents when exposed to malignant or otherhyperproliferative cells and can reduce hypercalcemia associated withthe maligancy. This selectivity and specificity of action makes thehypocalcemic vitamin D compounds useful and preferred antihypercalcemicagents as well as safely inhibiting hyperproliferation and promotingmalignant or hyperplastic cell differentiation. The compounds of thepresent invention, thus, overcome the shortcomings of the known activevitamin D₃ compounds described above, and can be considered preferredagents for the control and treatment of malignant diseases such breast,prostate, testicular and colon cancer, as well as other neoplasms suchas pancreatic cancer, endometrial cancer, small cell and non-small cellcancer of the lung (including squamous, adneocarcinoma and large celltypes), squamous cell of the head and neck, bladder, ovarian andcervical cancers, myeloid and lymphocyltic leukemia, lymphoma, hepatictumors, medullary thyroid carcinoma, multiple myeloma, melanomaretinoblastoma, and sarcomas of the soft tissue and bone, i.e. neoplasmsthat express vitamin D receptors.

[0037] Suitable hypocalcemic vitamin D compounds in accordance with thepresent invention include: 1α,24-dihydroxyvitamin D₂,1α,24-dihydroxyvitamin D₄, 1α,25-dihydroxyvitamin D₂,1α,25-dihydroxyvitamin D₄, 1α-hydroxyvitamin D₂, and 1α-hydroxyvitaminD₄. Among those compounds of formula (I) that have a chiral center inthe sidechain, such as at C-24, it is understood that both epimers(e.g., R and S) and the racemic mixture are within the scope of thepresent invention.

[0038] Thus, the present invention provides a method of treatinghypercalcemia associated with malignant cells with an effective amountof a hypocalcemic vitamin D compound. The effective dosage amount on adaily basis per kilogram of body weight of the patient ranges from about0.01 μg/kg/day to about 2.0 μg/kg/day.

[0039] The compounds of formula (I) can be prepared as described, e.g.,in U.S. Pat. No. 5,488,120 issued to Knutson et al., U.S. Pat. Nos.4,670,190 and 4,554,106 issued to DeLuca et al., U.S. Pat. No. 5,486,636issued to DeLuca et al., and Strugnell et al., 310 Biochem. J. (1995)pp. 233-241, all of which are incorporated herein by reference.

[0040] The biopotencies of the compounds of formula (I) have beenstudied and compared to that of 1α,25-dihydroxyvitamin D₃, the activehormonal form of vitamin D and the standard against which all vitamin Dcompounds and analogues are measured. For example, it has been foundthat the vitamin D receptor (VDR) binding affinities of the compounds offormula (I), or their active metabolites, are substantially equivalentto (i.e., equal to or up to 3 times weaker than) the affinity of1α,25-dihydroxyvitamin D₃. Such receptor binding affinities areindicative of potent biological activity.

[0041] At the same time, it has been found that compounds of formula (I)are significantly less toxic than their corresponding vitamin D₃analogues. For example, in parent co-pending application, Ser. No.08/265,438, the disclosure of which is incorporated herein by reference,the LD₅₀ for 1α-hydroxyvitamin D₄ was found to be 1.0 mg/kg in males and3.0 mg/kg in females, i.e., substantially less toxic than1α-hydroxyvitamin D₃ (LD₅₀˜0.2 mg/kg). Further, in the parent U.S. Pat.No. 5,403,831, and its grandparent U.S. Pat. No. 5,104,864, both ofwhich are incorporated herein by reference, it has been shown that1α-hydroxyvitamin D₂ has the same biopotency as 1α-hydroxyvitamin D₃ and1α,25-dihydroxyvitamin D₃ but is much less toxic. Even dosages up to 10μg/day of 1α-hydroxyvitamin D₂ in women with postmenopausal osteoporosiselicited only mild hypercalciuria (U.Ca>300 mg/24 hrs), and no markedhypercalcemia (S. Ca>1 1.0 mg/dL) solely due to 1α-hydroxyvitamin D₂ wasevident. Additionally, the compound did not adversely affect kidneyfunction, as determined by creatinine clearance and BUN; nor did itincrease urinary excretion of hydroxyproline, indicating the absence ofany stimulatory effect on bone resorption. Administration of1α-hydroxyvitamin D₂ to healthy adult males in dosages up to 8 μg/dayshowed no clinically significant hypercalcemia or other adverse effects.

[0042] The hypocalcemic vitamin D compounds of the present invention areuseful as active compounds in pharmaceutical compositions having reducedside effects and low toxicity as compared with the known analogues ofactive forms of vitamin D₃.

[0043] The pharmacologically active compounds of this invention can beprocessed in accordance with conventional methods of pharmacy to producemedicinal agents for administration to patients, e.g., mammals includinghumans. For example, the hypocalcemic vitamin D compounds can beemployed in admixtures with conventional excipients, e.g.,pharmaceutically acceptable carrier substances suitable for enteral(e.g., oral), parenteral or topical application which do notdeleteriously react with the active compounds.

[0044] Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions, alcohols, gum arabic, vegetable oils(e.g., almond oil, corn oil, cottonseed oil, peanut oil, olive oil,coconut oil), mineral oil, fish liver oils, oily esters such asPolysorbate 80, polyethylene glycols, gelatine, carbohydrates (e.g.,lactose, amylose or starch), magnesium stearate, talc, silicic acid,viscous paraffin, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinylpyrrolidone, etc.

[0045] The pharmaceutical preparations can be sterilized and, ifdesired, be mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, coloring, flavoring and/or one ormore other active compounds, for example, vitamin D₃ and its1α-hydroxylated metabolites, conjugated estrogens or their equivalents,anti-estrogens, calcitonin, biphosphonates, calcium supplements,cobalamin, pertussis toxin and boron.

[0046] For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solution, as well assuspensions, emulsions, or implants, including suppositories. Parenteraladministration suitably includes subcutaneous, intramuscular, orintravenous injection, nasopharyngeal or mucosal absorption, ortransdermal absorption. Ampoules are convenient unit dosages.

[0047] For enteral application, particularly suitable are tablets,dragees, liquids, drops, suppositories, lozenges, powders, or capsules.A syrup, elixir, or the like can be used if a sweetened vehicle isdesired.

[0048] For topical application, suitable nonsprayable viscous,semi-solid or solid forms can be employed which include a carriercompatible with topical application and having a dynamic viscositypreferably greater than water, for example, mineral oil, almond oil,self-emulsifying beeswax, vegetable oil, white soft paraffin, andpropylene glycol. Suitable formulations include, but are not limited to,creams, ointments, lotions, solutions, suspensions, emulsions, powders,liniments, salves, aerosols, transdermal patches, etc., which are, ifdesired, sterilized or mixed with auxiliary agents, e.g., preservatives,stabilizers, demulsifiers, wetting agents, etc. A cream preparation inaccordance with the present invention suitably includes, for example,mixture of water, almond oil, mineral oil and self-emulsifying beeswax;an ointment preparation suitably includes, for example, almond oil andwhite soft paraffin; and a lotion preparation suitably includes, forexample, dry propylene glycol.

[0049] Topical preparations of the compound in accordance with thepresent invention useful for the treatment of skin disorders may alsoinclude epithelialization-inducing agents such as retinoids (e.g.,vitamin A), chromanols such as vitamin E, β-agonists such asisoproterenol or cyclic adenosine monophosphate (cAMP),anti-inflammatory agents such as corticosteroids (e.g., hydrocortisoneor its acetate, or dexamethasone) and keratoplastic agents such as coaltar or anthralin. Effective amounts of such agents are, for example,vitamin A about 0.003 to about 0.3% by weight of the composition;vitamin E about 0.1 to about 10%; isoproterenol about 0.1 to about 2%;cAMP about 0.1 to about 1%; hydrocortisone about 0.25 to about 5%; coaltar about 0.1 to about 20%; and anthralin about 0.05 to about 2%.

[0050] For rectal administration, the compound is formed into apharmaceutical composition containing a suppository base such as cacaooil or other triglycerides. To prolong storage life, the compositionadvantageously includes an antioxidant such as ascorbic acid, butylatedhydroxyanisole or hydroquinone.

[0051] For treatment of hypercalcemia associated with maligancy, oraladministration of the pharmaceutical compositions of the presentinvention is preferred. Generally, the compound of this invention isdispensed by unit dosage form comprising about 0.5 μg to about 25 μg ina pharmaceutically acceptable carrier per unit dosage. The dosage of thecompound according to this invention generally is about 10 μg to 200μg/day.

[0052] For topical treatment of skin disorders, the dosage of thecompound of the present invention in a topical composition generally isabout 0.01 μg to about 50 μg per gram of composition. For treatment ofskin cancers, the dosage of the hypocalcemic vitamin D compound in alocally applied composition generally is about 0.01 μg to 100 μg pergram composition.

[0053] It is noted that dosing of the hypocalcemic compounds inaccordance with the present invention can also be done on an episodicbasis, in which case higher doses can be used generally about 20 μg toabout 200 μg given once every 2 to 7 days. The dose can be given as asingle dose or a divided dose in 2 to 5 subdoses, the subdoses given,e.g., one every hour until the total dose is taken.

[0054] Those of ordinary skill in the art will readily optimizeeffective doses and coadministration regimens as determined by goodmedical practice and the clinical condition of the individual patient.Regardless of the manner of administration, it will be appreciated thatthe actual preferred amounts of active compound in a specific case willvary according to the efficacy of the specific compound employed, theparticular compositions formulated, the mode of application, and theparticular situs and organism being treated. For example, the specificdose for a particular patient depends on age, body weight, general stateof health, on diet, on the timing and mode of administration, on therate of excretion, and on medicaments used in combination and theseverity of the particular disorder to which the therapy is applied.Dosages for a given host can be determined using conventionalconsiderations, e.g., by customary comparison of the differentialactivities of the subject compounds and of a known agent, such as bymeans of an appropriate conventional pharmacological protocol.

[0055] Further, included within the scope of the present invention isthe co-administration of a hypocalcemic vitamin D compound with aanticancer agent, e.g., a cytotoxic agent, Such agents suitably includeantimetabolites (e.g., 5-fluoro-uracil, methotrexate, fludarabine),antimicrotubule agents (e.g., vincristine, vinblastine, taxanes such aspaclitaxel, docetaxel), an alkylating agent (e.g., cyclophasphamide,melphalan, biochoroethylnitrosurea, hydroxyurea), platinum agents (e.g.cisplatin, carboplatin, oxaliplatin, JM-216, CI-973), anthracyclines(e.g., doxrubicin, daunorubicin), antibiolitics (e.g., mitomycin,idarubicin, adriamycin, daunomycin), topoisomerase inhibitors (e.g.,etoposide, camptothecins) or any other antineoplastic agents.(estramustine phosphate, prednimustine). It is anticipated that thehypocalcemic vitamin D compounds used in combination with variousanticancer drugs can give rise to a significantly enhanced cytotoxiceffect on cancerous cells, thus providing an increased therapeuticeffect. Specifically, as a significantly increased growth-inhibitoryeffect is obtained with the above disclosed combinations utilizing lowerconcentrations of the anticancer drugs compared to the treatment regimesin which the drugs are used alone, there is the potential to providetherapy wherein adverse side effects associated with the anticancerdrugs are considerably reduced than normally observed with theanticancer drugs used alone in larger doses. Possible dose ranges ofthese co-administered anticancer agents are about 0.1 to 20 mg/kg/day.

[0056] The term “co-administration” is meant to refer to anyadministration route in which two or more agents are administered to apatient or subject. For example, the agents may be administeredtogether, or before or after each other. The agents may be administeredby different routes, e.g., one agent may be administered intravenouslywhile the second agent is administered intramuscularly, intravenously ororally. The agents may be administered simultaneously or sequentially,as long as they are given in a manner sufficient to allow both agents toachieve effective concentrations in the body. The agents also may be inan admixture, as, for example, in a single tablet. In sequentialadministration, one agent may directly follow administration of theother or the agents may be give episodically, i.e., one can be given atone time and the other at a later time, typically within a week. Anexample of a suitable co-administration regimen is where a hypocalcemicvitamin D compound is administered from 0.5 to 7 days prior toadministration of a cytotoxic agent.

[0057] Also included within the scope of the present invention is theco-administration of effective dosages of the analogue of formula (I) inconjunction with administration of hormones or other agents, e.g.,estrogens, which are known to ameliorate bone diseases or disorders. Asnoted above, prostate cancer often metastasizes to bone, causing boneloss and associated pain. Such bone agents may include conjugatedestrogens or their equivalents, calcitonin, bisphosphonates, calciumsupplements, cobalamin, pertussis toxin and boron. It is contemplatedthat these bone agents also have an antihypercalcemic effect and mayenhance the treatment of malignancy-associated hypercalcemia. Possibledose ranges for these co-administered bone agents are provided inTable 1. TABLE 1 Possible Oral Dose Ranges for Various Bone AgentsCo-Administered With 1α-Hydroxyvitamin D of Formula (I) Dose Ranges MostAgent Broad Preferred Preferred Conjugated Estrogens or 0.3-5.0 0.4-2.40.6-1.2 Equivalent (mg/day) Sodium Fluoride (mg/day)  5-150 30-75 40-60Calcitonin (IU/day)  5-800  25-500  50-200 Bisphosphonates (mg/day)0.5-20   1-15  5-10 Calcium Supplements  250-2500  500-1500  750-1000(mg/day) Cobalamin (μg/day)  5-200  20-100 30-50 Pertussis Toxin(mg/day)   0.1-2000  10-1500  100-1000 Boron (mg/day)  0.10-3000  1-250 2-100

[0058] The present invention is further explained by the followingexamples which should not be construed by way of limiting the scope ofthe present invention.

VDR BINDING ANALYSES EXAMPLE 1 1α, 24-dihydroxyvitamin D₂[1α,24-(OH)₂D₂]

[0059] The affinity of 1α,24-(OH)₂D₂ for the mammalian vitamin Dreceptor (VDR) was assessed using a commercially available kit of bovinethymus VDR and standard 1,25-(OH)₂D₃ solutions from Incstar (Stillwater,Minn.). The half-maximal binding of chemically synthesized 1α,24-(OH)₂D₂was approximately 150 pg/ml whereas that of 1α,25-(OH)₂D₃ was 80 pg/ml.Thus, the 1α,24-(OH)₂D₂ had a very similar affinity for bovine thymusVDR as did 1α,25-(OH)₂D₃, indicating that 1α,24-(OH)₂D₂ has potentbiological activity.

EXAMPLE 2 1α,24-dihydroxy Vitamin D₄ [1α,24-(OH)₂D₄]

[0060] The VDR affinity binding of 1α,24-(OH)₂D₄ was investigated. The1α,24-(OH)₂D₄ was incubated with vitamin D receptor and radiolabeledtracer 1α,25-(OH)₂D₃. After incubation, the amount of radioactivitybound to the receptor was determined and compared with the amount boundafter co-incubation of unlabeled and labeled 1α,25-(OH)₂D₃. It was foundthat 50 pg/tube of 1α,24-(OH)₂D₄ was equivalent to approximately 20 pg1α,25-(OH)₂D₃.

[0061] These results show that 1α,24-(OH)₂D₄ binds slightly less tightlyto the vitamin D receptor than does 1α,25-(OH)₂D₃. Such data mean that1α,24-(OH)₂D₄ has high affinity for the VDR and significant biologicalactivity, similar to that of 1α,25-(OH)₂D₃. These data are consistentwith gene expression studies done (described below) with 1α,24-(OH)₂D₄which demonstrate that 1α,24-(OH)₂D₄ is only slightly less active thanis 1α,25-(OH)₂D₃.

[0062] These results are surprising and unexpected in view of the priorart. They are contrary to the normative wisdom in the vitamin D artregarding the very low degree of biological activity of vitamin D₄compounds.

EXAMPLE 3 1α,24-dihydroxyvitamin D₂ [1α,24-(OH)₂D₂]

[0063] VDR binding of vitamin D compounds by prostate cells isdemonstrated using the techniques of Skowronski et al., 136Endocrinology (1995) 20-26, which is incorporated herein by reference.Prostate-derived cell lines are cultured to near confluence, washed andharvested by scraping. Cells are washed by centrifugation, and the cellpellet resuspended in a buffered salt solution containing proteaseinhibitors. The cells are disrupted by sonication while cooling on ice.The supernatant obtained from centrifuging the disrupted cells at207,000×g for 35 min at 4EC is assayed for binding. 200 TL of solubleextract, (1-2 mg protein/ml supernatant) is incubated with a 1 nM³H-1α,25-(OH)₂D₃ and increasing concentrations of 1α,24-(OH)₂-D₂(0.01-100 nM) for 16-20 hr at 4EC. Bound and free hormones are separatedwith hydroxylapatite using standard procedures. Specific binding iscalculated by subtracting nonspecific binding obtained in the presenceof a 250-fold excess of nonradioactive 1α,25-(OH)₂D₃ from the totalbinding measured. The results demonstrate that 1α,24-(OH)₂D₂ has strongaffinity for prostate VDR, indicating that 1α,24-(OH)₂D₂ has potentbiological activity in respect of prostate cells.

EXAMPLE 4 1α,24-dihydroxy vitamin D₄ [1α,24-(OH)₂D₄]

[0064] The procedure of Example 3 is repeated using the active vitamin Danalogue 1α,24-(OH)₂D₄, and the specific binding is determined. Theresults demonstrate that 1α,24-(OH)₂D₄ has strong affinity for prostateVDR, indicating that 1α,24-(OH)₂D₄ has potent biological activity inrespect of prostate cells.

EXAMPLE 5 1α,25-dihydroxyvitamin D₄ [1α,25-(OH)₂D₄]

[0065] The procedure of Example 3 is repeated using the active vitamin Danalogue 1α,25-(OH)₂D₄, and the specific binding is determined. Theresults demonstrate that 1α,25-(OH)₂D₄ has strong affinity for prostateVDR, indicating that 1α,25-(OH)₂D₄ has potent biological activity inrespect of prostate cells.

GENE EXPRESSION EXAMPLE 6 1α,24-dihydroxy vitamin D₄ [1α,24-(OH)₂D₄]

[0066] Using the plasmids p(CT4)⁴TKGH, a vitamin D receptor(VDR)-expressing plasmid, and pSG5-hVDR1/3, a plasmid containing aGrowth Hormone (GH) gene, under the control of a vitamin D-responsiveelement (VDRE), experiments were conducted to explore the ability of1α,24-(OH)₂D₄ to induce vitamin D-dependent growth hormone acting as areporter gene compared to that of 1α,25-(OH)₂D₃. Cells in culture weretransfected with these two plasmids. One plasmid contained the gene forGrowth Hormone (GH) under the control of the vitamin D responsiveelement (VDRE) and the other plasmid contained the structural gene forthe vitamin D receptor (VDR). These transfected cultures were incubatedwith 1α,24-(OH)₂D₄ or 1α,25-(OH)₂D₃, and the production of growthhormone was measured. Table 2 below shows the results of this assay:TABLE 2 Induction of Growth Hormone by Vitamin D Compounds ConcentrationGrowth Hormone Compound Used (M) Induction (ng/ml) 1,25-(OH)₂D₃ 1 ×10⁻¹⁰ 39 1,25-(OH)₂D₃ 5 × 10⁻¹⁰ 248 1,24-(OH)₂D₄ 5 × 10⁻¹⁰ 1651,24-(OH)₂D₄ 1 × 10⁻⁹  628 1,24-(OH)₂D₄ 5 × 10⁻⁹  1098

[0067] These data show that the ability of 1α,24-(OH)₂D₄ to stimulatevitamin D-dependent growth hormone is nearly equivalent to that of1α,25-(OH)₂D₃. Such results are truly surprising and would not have beenexpected by following the teachings of the prior art.

EXAMPLE 7 1α,24(S)-dihydroxyvitamin D₂ and 1α,24(R)-dihydroxy-vitamin D₂[1α,24(S)-(OH)₂D₂ and 1α,24(R)-(OH)₂D₂]

[0068] The gene expression study described in Example 6 was conducted tocompare the biological activity in vitro of chemically synthesized1α,24(S)-(OH)₂D₂ and 1α,24(R)-(OH)₂D₂, with 1α,25-(OH)₂D₃ and 25-OH-D₃.The vitamin D-dependent transcriptional activation model system was usedin which plasmids pSG5-hVDR1/3 and p(CT4)⁴TKGH were co-transfected intoGreen monkey kidney, COS-1 cells.

[0069] Transfected cells were incubated with vitamin D metabolites andgrowth hormone production was measured. As shown in Table 3, both1α,24(S)-(OH)₂D₂ and its epimer, 1α,24(R)-(OH)₂D₂, had significantlymore activity in this system than 25-OH-D₃, with 1α,24(S)-(OH)₂D₂ havingnearly the same activity as 1α,25-(OH)₂D₃. TABLE 3 Vitamin D-InducibleGrowth Hormone Production In Transfected COS-1 Cells Vitamin DClnducibleGrowth Hormone Production Net vitamin Total GH DCinducible MolarProduction* GH-production Inducer Concentration (ng/ml) (ng/ml) Ethanol44 0 25-OH-D₃ 1 × 10⁻⁷  245 201 1 × 10⁻⁶  1100 1056 1 × 10⁻⁵  775 7311α,25-(OH)₂D₃ 1 × 10⁻¹⁰ 74 30 1 × 10⁻⁹  925 881 1 × 10⁻⁸  1475 14411α,24(S)-(OH)₂D₂ 5 × 10⁻¹⁰ 425 381 5 × 10⁻⁹  1350 1306 5 × 10⁻⁸  11821138 1α,24(R)-(OH)₂D₂ 1 × 10⁻⁹  80 36 1 × 10⁻⁸  1100 1056 1 × 10⁻⁷  13001256

INHIBITION OF CELL PROLIFERATION EXAMPLE 8 1α,24-dihydroxyvitamin D₂[1α,24-(OH)₂D₂]

[0070] Inhibition of cell proliferation is demonstrated using thetechniques of Skowronski et al., 132 Endocrinology (1993) 1952-1960 and136 Endocrinology (1995) 20-26, both of which are incorporated herein byreference. The cell lines, LNCaP and PC-3, which are derived from humanprostate adenocarcinoma, are seeded in six-well tissue culture plates ata density of about 50,000 cells/plate. After the cells have attached andstabilized, about 2-3 days, the medium is replenished with mediumcontaining vehicle or the active vitamin D analogue 1α,24-(OH)₂D₂, atconcentrations from 10⁻¹¹ M to 10⁻⁷ M. Medium containing test analogueor vehicle is replaced every three days. After 6-7 days, the medium isremoved, the cells are rinsed, precipitated with cold 5% trichloroaceticacid, and washed with cold ethanol. The cells are solubilized with 0.2 Nsodium hydroxide, and the amount of DNA determined by standardprocedures. The results show that cultures incubated with 1α,24-(OH)₂D₂in accordance with the present invention have significantly fewer cellsthan the control cultures.

EXAMPLE 9 1α,24-dihydroxy Vitamin D₄ [1α,24-(OH)₂D₄]

[0071] The procedure of Example 8 is repeated using the active vitamin Danalogue 1α,24-(OH)₂D₄, and the cell number is determined. Culturesincubated with 1α,24-(OH)₂D₄ have significantly fewer cells than thecontrol cultures.

EXAMPLE 10 1α,25-dihydroxyvitamin D₄ [1α,25-(OH)₂D₄]

[0072] The procedure of Example 8 is repeated using the active vitamin Danalogue 1α,25-(OH)₂D₄, and the cell number is determined. Culturesincubated with 1α,25-(OH)₂D₄ have significantly fewer cells than thecontrol cultures.

STIMULATION OF CELL DIFFERENTIATION EXAMPLE 11 1α,24-dihydroxyvitamin D₂[1α,24-(OH)₂D₂]

[0073] Using the techniques of Skowronski et al., 132 Endocrinology(1993) 1952-1960 and 136 Endocrinology (1995) 20-26, both of which areincorporated herein by reference, cells of the cell line, LNCaP, whichis derived from a human metastatic prostate adenocarcinoma and known toexpress PSA, are seeded in six-well tissue culture plates at a densityof about 50,000 cells/plate. After the cells have attached andstabilized, about 2-3 days, the medium is replenished with mediumcontaining vehicle or the active vitamin D analogue, 1α,24-(OH)₂D₂, atconcentrations from 10⁻¹¹ M to 10⁻⁷ M. After 6-7 days, the medium isremoved and stored at −20EC for prostate specific antigen (PSA)analysis.

[0074] The cells from parallel cultures are rinsed, precipitated, andthe amount of DNA determined by standard procedures. PSA is measured bystandard known methods. Cultures incubated with 1α,24-(OH)₂D₂ havesignificantly more PSA than control cultures when expressed as mass ofPSA/cell.

EXAMPLE 12 1α,24-dihydroxyvitamin D₄ [1α,24-(OH)₂D₄]

[0075] The procedure of Example 12 is repeated except the active vitaminD analogue is 1α,24-(OH)₂D₄. The PSA is measured and cultures incubatedwith 1α,24-(OH)₂D₄ have significantly more PSA than control cultureswhen expressed as mass of PSA/cell.

EXAMPLE 13 1α,25-dihydroxyvitamin D₄ [1α,24-(OH)₂D₄]

[0076] The procedure of Example 12 is repeated except the active vitaminD analogue is 1α,25-(OH)₂D₄. The PSA is measured and cultures incubatedwith 1α,25-(OH)₂D₄ have significantly more PSA than control cultureswhen expressed as mass of PSA/cell.

CLINICAL STUDIES EXAMPLE 14 General Treatment of MAH

[0077] Patients with malignancy-associated hypercalcemia participate inan open-label study of a hypocalcemic vitamin D compound in accordancewith the present invention. Patients are restricted to daily calciumintake of about 400-500 mg. Each patient is also asked to drink 4-6 cupsof fluid more than usual intake to assure adequate oral hydration.

[0078] Each subject is monitored at regular intervals for: (1)hypercalcemia, serum PTHrP levels, hyperphosphatemia, hypercalciuria,hyperphosphaturia and other toxicity; (2) evidence of changes in theprogression of metastatic disease; and (3) compliance with theprescribed test drug dosage.

[0079] The dosing regimen is typically on a daily dose basis of 10 μg or20 μg per day to about 100 μg/day for 10 weeks. Alternatively, anon-daily dosing regimen can be used, e.g., 40 μg given every other day,100 μg given once a week. The route of administration can vary from oralto intravenous to regional delivery (e.g., arterial infusion, via theportal vein). Oral is, of course, the easiest and most cost effectiveroute. Regional delivery permits high dosing and generally avoids anyproduction of hypercalcemia. Although, in the case of the compound ofthe present invention, the compound is substantially hypocalcemic.

[0080] After the treatment period, CAT, scans, X-rays and bone scansused for evaluating the progress of metastatic disease show stabledisease or partial remission in many patients treated at the lowerdosage, and stable disease and partial or complete remission in manypatients treated at the higher dosage. Serum calcium levels are in thenormal range and serum levels of PTHrP are redeuced.

EXAMPLE15 Treatment of MAH Using 1α,24(s)-dihydroxyvitamin D₂[1α,24(S)-(OH)₂D₂]

[0081] The procedure of example 14 is carried out using 1α,24-(OH)₂D₂.The results show serum calcium levels in the normal range and serumlevels of PTHrP reduced.

EXAMPLE 16 Treatment of MAH Using 1α-hydroxyvitamin D₂ [1α-OH-D₂]

[0082] The procedure of example 14 is carried out using 1α-OH-D₂. Theresults show serum calcium in the normal range and serum PTHrP levelsreduced.

[0083] While the present invention has now been described andexemplified with some specificity, those skilled in the art willappreciate the various modifications, including variations, additions,and omissions, that may be made in what has been described. Accordingly,it is intended that these modifications also be encompassed by thepresent invention and that the scope of the present invention be limitedsolely by the broadest interpretation lawfully accorded the appendedclaims.

What is claimed is:
 1. A method of treating hypercalcemia associated with malignant or neoplastic cells, comprising treating the cells with an effective amount of a hypocalcemic vitamin D compound having a hydrocarbon moiety at the C₂₄ position.
 2. The method of claim 1, wherein the cells are cancers of the breast, colon, lung, neck and head, pancreas, endometrium, bladder, cervix, testes, ovaries, squamous cell carcinoma, myeloid and lymphocytic leukemia, lymphoma, medullary thyroid carcinoma, melanoma, multiple myeloma, retinoblastoma or sarcomas of the soft tissues and bone.
 3. The method of claim 1, wherein the hypocalcemic vitamin D is a compound represented by formula (I)

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R¹ and R² are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹ and R² cannot both be an alkenyl group, or taken together with the carbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes a bond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or, taken with R¹ or R², constitutes a double bond, and X³ is hydrogen or hydroxyl provided that at least one of X¹, X² and X³ is hydroxyl; and Y is a methylene group if the bond to Y is a double bond or is a methyl group or hydrogen if the bond to Y is a single bond.
 4. The method of claim 1, wherein said hypocalcemic vitamin D is a 1α-hydroxvitamin D compound is represented by formula (I)

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R¹ and R² are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹ and R² cannot both be an alkenyl group, or taken together with the carbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes a bond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or, taken with R¹ or R², constitutes a double bond.
 5. The method of claim 4, wherein the compound of formula (I) is 1α,24-dihydroxyvitamin D₂, 1α,24-dihydroxyvitamin D₄, 1α,25-dihydroxyvitamin D₂, 1α,25-dihydroxyvitamin D₄, 1α-hydroxyvitamin D₂ or 1α-hydroxyvitamin D₄.
 6. A method in accordance with claim 1, wherein a dosing regimen for the hypocalcemic vitamin D compound is a daily regimen or an episodic regimen.
 7. A method in accordance with claim 6, wherein the espisodic regimen is a dose once every 2 to 7 days.
 8. A method in accordance with claim 6, wherein the hypocalcemic vitamin D compound is administered daily at a dose of about 10 to 100 μg/day.
 9. A method in accordance with claim 6, wherein the hypocalcemic vitamin D compound is orally, intravenously or regionally delivered to a cancer site.
 10. A method in accordance with claim 9, wherein the hypocalcemic vitamin D compound is administered orally.
 11. A method in accordance with claim 1, wherein the hypocalcemic vitamin D compound is co-administered with a cytotoxic agent.
 12. A method in accordance with claim 11, wherein the cytotoxic agent is an antimetabolite, and antimicrotubule agent, an alkyating agent, a platinum agent, an anthracycline, a topoisomase inhibitor, or an antibiotic.
 13. A method in accordance with claim 12, wherein the antimetabolite is 5-fluoro-uracil, methotrexate or fludarabine.
 14. A method in accordance with claim 12, wherein the antimicrotubule agent is vincristine, vinblastine or a taxane.
 15. A method in accordance with claim 14, wherein the taxane is paclitaxel or docetaxel.
 16. A method in accordance with claim 12, wherein the alkylating agent is cyclophasphamide, melphalan, biochoroethylnitrosurea or hydroxyurea.
 17. A method in accordance with claim 12, wherein the platinum agent is cisplatin, carboplatin, oxaliplatin, JM-216 or CI-973.
 18. A method in accordance with claim 12, wherein the anthracycline is doxrubicin or daunorubicin.
 19. A method in accordance with claim 12, wherein the antibiotic is mitomycin, idarubicin, adriamycin or daunomycin.
 20. A method in accordance with claim 12, wherein the topoisomerase inhibitior is etoposide or camptothecins.
 21. A method in accordance with claim 12 wherein the cytotoxic agent is estramustine phosphate or prednimustine.
 22. A method of treating a human to alleviate hypercalcemia associated with breast cancer, colon cancer, prostate cancer, testicular cancer, pancreatic cancer, endometrial cancer, small cell and non-small cell cancer of the lung (including squamous, adneocarcinoma and large cell types), squamous cell of the head and neck, bladder, ovarian and cervical cancers, myeloid and lymphocyltic leukemia, lymphoma, hepatic tumors, medullary thyroid carcinoma, multiple myeloma, melanoma, retinoblastoma or sarcomas of the soft tissue and bone, comprising administering to the human therapeutic amount of a hypocalcemic vitamin D compound.
 23. A method of claim 22, wherein said hypocalcemic vitamin D is a 1α-hydroxyvitamin D compound represented by formula (III)

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R¹ and R² are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹ and R² cannot both be an alkenyl group, or taken together with the carbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes a bond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or, taken with R¹ or R², constitutes a double bond.
 24. The method of claim 23, wherein said therapeutic amount is 0.01 μg/kg/day to 2.0 μg/kg/day.
 25. The method of claim 23, wherein the compound of formula (I) is 1α,24-dihydroxyvitamin D₂, 1α,24-dihydroxyvitamin D₄, 1α,25-dihydroxyvitamin D₂, 1(1,25-dihydroxyvitamin D₄, 1α-hydroxyvitamin D₂ or 1α-hydroxyvitamin D₄.
 26. A method of treating a human to alleviate hypercalcemia associated with malignant cells, comprising administering to the patient a hypocalcemic vitamin D compound, and a cytotoxic agent.
 27. A method in accordance with claim 26, wherein the hypocalcemic vitamin D compound is administered from 0.5 to 7 days prior to administration of the cytotoxic agent.
 28. A method in accordance with claim 26, wherein the hypocalcemic vitamin D compound is administered 2 to 4 days prior to administration of the cytotoxic agent.
 29. A method of claim 26, wherein said hypocalcemic vitamin D is a 1α-hydroxyvitamin D compound represented by formula (III)

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R¹ and R² are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹ and R² cannot both be an alkenyl group, or taken together with the carbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes a bond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or, taken with R¹ or R², constitutes a double bond.
 30. The method of claim 29, wherein the therapeutic amount is 0.01 μg/kg/day to 2.0 μg/kg/day.
 31. The method of claim 29, wherein the compound of formula (I) is 1α,24-dihydroxyvitamin D₂, 1α,24-dihydroxyvitamin D₄, 1α,25-dihydroxyvitamin D₂, 1α,25-dihydroxyvitamin D₄, 1α-hydroxyvitamin D₂ or 1α-hydroxyvitamin D₄.
 32. A method in accordance with claim 29, wherein the cytotoxic agent is an antimetabolite, and antimicrotubule agent, an alkyating agent, a platinum agent, an anthracycline, a topoisomase inhibitor, or an antibiotic.
 33. A method of lowering serum parathyroid hormone related protein in a human patient by administering to the human an effective amount of a hypocalcemic vitamin D compound. 